South African Journal of Botany 72 (2006) 232 – 237 www.elsevier.com/locate/sajb

Antibacterial, antifungal and antitubercular activity of (the roots of) Pelargonium reniforme (CURT) and Pelargonium sidoides (DC) () root extracts

S.P.N. Mativandlela, N. Lall *, J.J.M. Meyer

Department of Botany, University of Pretoria, Pretoria 0002, South Africa

Received 10 May 2005; accepted 26 August 2005

Abstract

Root extracts of Pelargonium reniforme CURT and Pelargonium sidoides DC were evaluated for antibacterial and antifungal assays using the agar dilution while antitubercular assays were done using the BACTEC method at concentrations ranging from 5Â103 to 500.0 mg/L. The ethanol and acetone extracts of the roots of P. sidoides inhibited the growth of Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae at a concentration of 5Â103 mg/L. Both acetone and ethanol extracts of P. reniforme and only the ethanol extract of P. sidoides inhibited the growth of Aspergillus niger and Fusarium oxysporum significantly at a concentration of 5Â103 mg/L. Growth of Rhizopus stolonifer was suppressed by the ethanol extract of P. reniforme and P. sidoides at 5Â103 and 1Â103 mg/L, respectively. Acetone, chloroform and ethanol extracts of P. reniforme showed activity against M. tuberculosis exhibiting a minimum inhibitory concentration of 5Â103 mg/L. D 2006 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Antibacterial; Antifungal; Extracts; Mycobacterium tuberculosis; Pelargonium

1. Introduction Africa use these species to treat coughs, diarrhoea and tuberculosis (Watt and Breyer-Brandwijk, 1962). The medic- The importance of Pelargonium species (Geraniaceae) is inally active ingredients are found in the bitter tasting roots of well documented (Watt and Breyer-Brandwijk, 1962; Hutch- the (Helmstadter, 1996). A commonly used medicine ings, 1996). The genus Pelargonium comprises more than 250 produced in Germany, named, FUmckaloabo_ originates from natural species of perennial small shrubs, which are limited in the roots of P. sidoides and P. reniforme (Helmstadter, 1996; their geographical distribution. About 80% of Pelargonium Kayser et al., 1998). This medicine is extensively used species are confined to the southern parts of Africa, while in Germany for bronchitis, antibacterial and antifungal infec- others occur in Australia, New Zealand and the Far East. These tions. Although this (UmckaloaboR)is species usually grow in short grassland and sometimes with successfully employed in modern phytotherapy in Europe to shrubs and trees on stony soil varying from sand to clay-loam, cure infectious diseases of the respiratory tract, the scientific shale or basalt. The plants are evergreen when cultivated, but basis of its remedial effect is still unclear (Kayser and die back in nature during droughts and winter (May to August) Kolodziej, 1995). (Van der Walt and Vorster, 1985). Bacteria, which are associated with either primary or Pelargonium reniforme CURT and Pelargonium sidoides secondary infections of bronchitis, are Streptococcus pneumo- DC are highly valued by traditional healers for their curative niae, Haemophilus influenzae and Moraxella catarrhalis. H. properties and they are well known to generations of Khoi/San influenzae, a Gram-negative bacterium, is an obligate human and Xhosa (South African tribes) traditional healers (Wagner parasite that is passed from person to person by way of the and Bladt, 1975). The Xhosa and the Zulu tribes of South respiratory route. M. catarrhalis, a Gram-negative bacterium, causes bronchitis and pneumonia in children and adults. S. * Corresponding author. pneumoniae, a Gram-positive bacterium, infects the upper E-mail address: [email protected] (N. Lall). respiratory tract and can cause pneumonia, also it can infect the

0254-6299/$ - see front matter D 2006 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2005.08.002 S.P.N. Mativandlela et al. / South African Journal of Botany 72 (2006) 232–237 233 lining of the brain-spinal cord (meningitis), bones (osteomy- respectively. Seider and Taylor (2004) investigated the two elitis), joints (arthritis), ears (otitis media) and sinuses (sinusitis species against rapidly growing mycobacteria (M. and bronchitis) (Benjamin et al., 1991). aurum and M. fortuitum, M. phlei, M. abscessus and M. Apergillus niger, Fusarium oxysporum and Rhizopus smegmatis). This is the first report on antitubercular activity stolonifer are some of the fungal pathogens that can affect of these plants extracted using various solvents such as the respiratory tract. A. niger, is a causative agent of pulmonary chloroform, acetone and ethanol against M. tuberculosis using diseases including aspergillosis, bronchial asthma and acute BACTEC radiometric method. allergic alveolitis. The fungus colonizes old tuberculosis or bronchiostatic cavities, in which it forms a large colony 2. Materials and methods (aspergilloma); or it may actually invades the lung tissue to produce haemorrhagic and necrotizing pneumonia (MacSween 2.1. Plant material and Whaley, 1992). F. oxysporum is responsible for fusariosis, skin infection, respiratory tract infections (tuberculosis and Roots of P. reniforme and P. sidoides were collected from bronchitis) and arthritis and produces a 76% mortality rate in Qwaqwa, a region in the Free State province of South Africa. hospitalised immunocompromised patients (Monier et al., Voucher specimens of P. reniforme (P 092558) and P. sidoides 1994). R. stolonifer causes mucorosis disease and it has been (P 092559) were deposited and identified at the H.G.W.J. reported that exposure to large numbers of Rhizopus spores can Schweickerdt Herbarium (PRU), University of Pretoria, South cause respiratory complications (Alexopoulos et al., 1996). Africa. Previously, researchers have reported antimicrobial activity of extracts of Pelargoniums and their constituents against a few 2.2. Preparation of extracts bacterial (Staphylococcus aureus, S. pneumoniae, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas Air-dried and powdered roots of P. reniforme and P. aeruginosa and H. influenzae), and fungal (Microsporum sidoides (300 g each) were extracted three times with 1 L of canis, M. gypseum, A. fumigatus, Mucor racemosus, R. acetone, chloroform and ethanol separately for 2 h at room nigricans) pathogens as well as opportunistic yeasts such as temperature. The extracts were filtered through Whatman No. Candida albicans, C. glabrata, C. krusei and Cryptococcus 1 filter paper and concentrated with a rotary vacuum neoformans (Kolodziej, 2000; Kolodziej et al., 2003; Latte´ and evaporator (Bu¨chi Laboratoriums, Technik AG, Germany) to Kolodziej, 2000). Plant extracts of P. reniforme and P. sidoides dryness at reduced pressure. For antibacterial and antifungal have not been tested against the fungal pathogens, A. niger, F. assays, acetone and ethanol extracts were dissolved in oxysporum, R. stolonifer and the Gram-negative bacteria M. acetone to a concentration of 5Â104 and 1Â105 mg/L, catarrhalis, which are indirectly responsible for secondary respectively. For the antitubercular assay, all (3) extracts were infections in cases of bronchitis and tuberculosis. In the present dissolved in dimethyl sulphoxide (DMSO) to a concentration study, we have investigated their antimicrobial activity against of 5Â105 mg/L. the bacteria and fungi mainly responsible for bronchitis. We have also confirmed the findings of other researchers on the 2.3. Microorganisms and in vitro antimicrobial assays antibacterial activity of these species against S. pneumoniae and H. influenzae. 2.3.1. Bacteria Tuberculosis (TB) kills approximately 2 million people The bacteria used in this investigation H. influenzae (UPM each year, the global epidemic is growing and becoming more 2), M. catarrhalis (UPM 4), and S. pneumoniae (UPM 9) were problematic. The breakdown in health services, the spread of clinical isolates which were obtained from the Department of HIV/AIDS and the emergence of multidrug-resistant strains of Pathology, University of Pretoria, South Africa. Cultures were Mycobacterium tuberculosis (MDR) TB are contributing to maintained on Colombia agar (Oxoid, Basingstoke, UK) slants the worsening impact of this disease. It is estimated that supplemented with 5% horse blood to form chocolate agar. For between 2002 and 2020, approximately a billion people will assays, organisms were subcultured once and incubated at be newly infected, more than 150 million people will get sick, 37 -C on Mueller-Hinton (MH), (BIOLAB, Merck, South and 36 million will die of TB. The current threat in TB Africa) agar for 24 h. treatment lies in the emergence of strains resistant to two of the best antitubercular drugs, isoniazid (INH) and rifampicin 2.3.2. Antibacterial assay (RIF). The current TB-treatment comprises of 3–4 drugs for a For the antibacterial assay, the minimum inhibitory con- period of 6–9 months (Bloom, 2002). Novel drugs are centrations (MIC which is defined as the lowest concentration required which can shorten this long-treatment period and of the extract that inhibits more than 99% of the bacterial target multidrug resistant strains of TB. Previous studies have population) of the acetone and ethanol extracts were deter- investigated the anti-TB and antimycobacterial activity of the mined by incorporating various amounts (5Â103,1Â103 and two Pelargonium species. Kolodziej (2000) and Kolodziej et 500.0 mg/L) of the extracts into chocolate agar in sterile bottles al. (2003) tested acetone extract of both plant species against and placed in a water bath (50 -C) to prevent solidification, M. tuberculosis using Alamar blue assay and acetone extracts then withdrawn into Petri dishes and left to solidify for 4 h. The of P. sidoides using the BACTEC radiometric system, bacterial colonies were transferred into the sterile screw-capped 234 S.P.N. Mativandlela et al. / South African Journal of Botany 72 (2006) 232–237 round tubes with glass beads to which 5 ml of the saline (0.9% BACTEC 12B vials containing 4 ml of 7H12 medium broth to w/v NaCl) was added for achieving McFarland No. 1 turbidity achieve the desired final concentrations of 5000.0, 2500.0, standard (108 CFU/ml). A hundred microlitres of each 1000.0 and 500.0 mg/L together with PANTA (Becton suspension was smeared on Petri dishes containing the extracts Dickinson and Company, Ferndale, South Africa), an antimi- and the chocolate agar. The plates (three replications) were crobial supplement. incubated at 37 -C for 24 h and antimicrobial activity was Control experiments showed that a final concentration of evaluated thereafter. Streptomycin sulphate (Sigma Chemical DMSO (1%) in the medium had no adverse effect on the Co., South Africa) was added to the chocolate agar plates (final growth of M. tuberculosis. Streptomycin, isoniazid, rifampicin concentrations of 500.0, 10.0 and 50.0 mg/L) and served as a and ethambutanol (Sigma Chemical Co., South Africa), were positive control. Three Petri dishes containing only 200 Al used as positive drug controls. A homogenous culture (0.1 ml) acetone mixed with chocolate agar served as negative controls. of M. tuberculosis, yielding 1Â104 to 1Â105 colony-forming The highest concentration of acetone (4%) did not affect the units per millilitre (CFU mlÀ 1), was inoculated in the vials growth of any of the organisms. containing the extracts as well as in the control vials (Heifets et al., 1985). Three extract-free vials were used as controls 2.3.3. Fungi (medium+1% DMSO): two vials (V1) were inoculated in the The fungal pathogens used in the study, A. niger (UPFC same way as the vials containing the extracts, and one (V2) was 13), F. oxysporum (UPFC 97) and R. stolonifer (UPFC 312) inoculated with a 1:100 dilution of the inoculum (1:100 were from culture collection at the Department of Microbiol- control) to produce an initial concentration representing 1% ogy and Plant Pathology, University of Pretoria, South Africa. of the bacterial population (1Â102 to 1Â103 CFU mlÀ 1). The Each fungus was maintained on Potato Dextrose Agar (PDA), MIC was defined as the lowest concentration of the extract that (BIOLAB, Merck, South Africa) for 7 days at T25 -C. inhibited >99% of the bacterial population. Mycobacterium growing in 7H12 medium containing 14C- 2.3.4. Antifungal assay labelled substrate (palmitic acid) use the substrate and 14 14 For the antifungal assay, the required amount of acetone and produced CO2. The amount of CO2 detected (reflecting ethanol extracts were added to sterile PDA in 5 ml Petri dishes the rate and amount of growth occurring in the sealed vial) is before congealing to yield final concentrations of 5Â103, expressed in terms of the growth index (GI) (Middlebrook et 1Â103 and 500.0 mg/L. PDA plates with acetone alone al., 1977). Inoculated bottles were incubated at 37 -C and each inoculated with fungi served as growth controls. Once the agar bottle was assayed everyday to measure GI, at about the same had solidified, a 5-mm plug of a 7-day-old fungal culture was hour(s) until cumulative results were interpretable. The placed in the centre of the Petri dish containing the extract- difference in the GI values of the last two days is designated amended and unamended PDA plates. The plates were sealed as DGI. The GI readings of the vials containing the test extracts with parafilm and placed in a 25 -C incubator. Fungal growth were compared with the control vials (V2). Readings were was measured on two diametric lines after 3, 6 and 9 days of taken until the control vials, containing a hundred times lower growth. Each treatment was replicated three times and results dilution of the inoculum than the test vials, reached a GI of 30 expressed as the mean of three replicates. The results of 6 days or more. If the DGI values of the vials containing the test growth was statistically analysed using analysis of variance extracts were less than the control vials, the population was (ANOVA) and comparison of means by Duncan’s Multiple reported to be susceptible to the compound. Each test was Range Test. The antifungal agent amphotericin B (Fluka, replicated three times. Germany) added to the agar plates (final concentration of 0.5, Whenever results suggested contamination (e.g. large, rapid 1.0 and 2.0 mg/L) served as a positive control. The highest increase in GI), bottles were inspected and the organisms were concentration of acetone (4%) did not affect any of the stained by Ziehl-Neelsen stain to determine whether the visible organisms. microbial growth was a mycobacterial organism (Kleeberg et al., 1980). With this stain, the bacilli appear as brilliantly 2.4. M. tuberculosis stained red rods against a deep sky-blue background. Organ- isms often have a beaded appearance because of their A drug-susceptible strain of M. tuberculosis, H37Rv polyphosphate content and unstained vacuoles (Joklik et al., obtained from American Type, MD, USA Culture Collection 1968). (ATCC, 27294), was used (to investigate the activity of the Since anecdotal evidence suggests the use of a combination plant extracts). of ethanol extracts of two Pelargonium species (1:1) combined ethanol and acetone root extracts from both species were 2.4.1. Antitubercular assay screened for antitubercular activity. The radiometric respiratory technique using the BACTEC system was used for susceptibility testing against M. tubercu- 3. Results and discussion losis as described previously (Lall and Meyer, 2001; Lall et al., 2003). Solutions of all the extracts were prepared in DMSO to It was found from the antibacterial assay that the ethanol obtain a concentration of 5Â105 mg/L and stored at 4 -C until and acetone extracts of P. sidoides and its combination (1:1 to used. Subsequent dilutions were made in DMSO and added to investigate additive effect) with P. reniforme was active at S.P.N. Mativandlela et al. / South African Journal of Botany 72 (2006) 232–237 235

5Â103 mg/L against H. influenzae, M. catarrhalis and S. positive bacteria such as S. aureus, Proteus vulgaris, Bacillus pneumoniae. Complete inhibition activity of three bacteria on cereus, and S. epidermidis (Lis-Balchin et al., 1998a, 2003). exposure to Streptomycin sulphate was observed at 10.0 mg/L. The acetone and ethanol root extracts of P. reniforme and Kayser and Kolodziej (1997), found moderate activity of P. ethanol root extract of P. sidoides showed activity against the sidoides against S. pneumoniae and H. influenzae at concen- fungal pathogens at a concentration of 5Â103 mg/L (Fig. 1a– trations of 7.5Â103 and 5Â103 mg/L, respectively, by ethanol b). Activity of amphotericin B was observed on each fungi at (70%) root extracts. There have been few reports of these 0.5 mg/L. Previous in vitro antifungal assays Latte´and bacterial organisms being susceptible to other plant extracts. Kolodziej (2000) had revealed that the aqueous acetone Christoph et al. (2001) found antibacterial activity of Austra- extracts of the roots of P. reniforme were less potent exhibiting lian from Melaleuca alternifolia (Cheel) and niaouli aMICof8Â103 mg/L against the filamentous fungi oil isolated from M. quinquenervia at 0.01 (%v vÀ 1) against M. (Aspergillus fumigatus, Rhizopus nigricans, Penicillium itali- catarrhalis. We found that acetone extracts of P. reniforme cum) and opportunistic yeasts tested. Lis-Balchin and Deans were not active against these bacteria at the highest concen- (1996) assessed the methanolic extracts of representative tration (5Â103 mg/L) tested, similar to the findings of Magama species and cultivars of Pelargonium for activity against 25 et al., 2002, when testing Euclea crispa. Essential oils of P. different species of bacteria and A. niger. All samples were graveolens were found to be inactive against Moraxella sp. active against at least 18 bacterial species and some were active (Lis-Balchin et al., 1998b). Gram-negative bacteria have been against all 25 species, although there was very poor antifungal found to be less susceptible to plant extracts in earlier studies action. Other plant extracts have been found to be antifungal done by other researchers (Kuhnt et al., 1994; Afolayan and against the fungi tested in this study. Chandrasekaran and Meyer, 1995). Essential oils from leaves of scented Pelargo- Venkatesalu (2004), investigated the water and methanol nium species such as P. graveolens, P. tomentosum, P. extracts of Syzygium jambolanum for antifungal activity odaratissimum, P. denticulatum and P. ficifolium have been against A. niger and R. stolonifer and the highest zones of found to possess good antibacterial activity against Gram- inhibition were recorded at 1Â103 and 500.0 mg/L, respec-

(a) 60 *a A.niger a 50 a F.oxysporum b R.stolonifer 40 b b bc bc c c 30 c c d d d d Growth (mm) 20 d

10 e 0 Ac/Control Ac/500.0 Ac/1000.0 Ac/5000.0 Et/500.0 Et/1000.0 Et/5000.0 Sample (concentration mg/L) (b) 60 *a a a A.niger a 50 a a F.oxysporum ab ab R.stolonifer a 40 ab b b b b b

30 c c

Growth (mm) 20 c 10

0 Ac/Control Ac/500.0 Ac/1000.0 Ac/5000.0 Et/500.0 Et/1000.0 Et/5000.0 Sample (concentration mg/L)

Fig. 1. Antifungal activity of (a) P. reniforme acetone and ethanol extract and (b) P. sidoides acetone and ethanol extract. Results are expressed as a mean of three replicates and are significantly different. *Values of the bars within the sample concentration not followed by the same letter are significantly different, P <0.01. Ac=acetone; Et=ethanol. 236 S.P.N. Mativandlela et al. / South African Journal of Botany 72 (2006) 232–237

Table 1 Acknowledgements Antitubercular activity of Pelargonium root extracts against the drug sensitive strain of Mycobacterium tuberculosis (H37Rv) determined by the BACTEC radiometric method Thanks to Dr, Bernard Fourie and the technical assistants of the Medical Research Council (Pretoria) and Mahdi Ziaratnia Plant species Sensitive strain for their assistance. The National Research Foundation a b MIC (mg/L) DGI values (mg/L) supported the research financially. Pelargonium reniforme (acetone) 5Â103 1.5T0.7 (Sc) P. reniforme (chloroform) 5Â103 0.5T0.7 (S) P. reniforme (ethanol) 5Â103 2.5T0.7 (S) References P. reniforme+P. sidoides (acetone) 5Â103 À1.0T2.8 (S) 3 T P. reniforme+P. sidoides (chloroform) 5Â10 1.0 0.0 (S) Afolayan, A.J., Meyer, J.J.M., 1995. Antibacterial activity of Helichrysum 3 T P. reniforme+P. sidoides (ethanol) 5Â10 1.5 0.7 (S) aureonitens (Asteraceae). Journal of Ethnopharmacology 47, 109–111. d T e P. sidoides (acetone) na 35.5 6.3 (R ) Alexopoulos, C.J., Mims, C.W., Blackwell, M., 1996. Introductory Mycology, T P. sidoides (ethanol) na 276.0 9.89 (R) 4th ednR John Wiley and Sons, New York, USA, pp. 868–869. T P. sidoides (chloroform) na 18.5 4.94 (R) Benjamin, D.C., Kadner, R.J., Volk, W.A., 1991. Essential of Medical T Streptomycin 4.0 À5.0 0.0 (S) Microbiology. (4th edn)J.B. Lippincott Company, Philadelphia, pp. 41–65. T Ethambutol 6.0 0.33 0.0 (S) Bloom, B.R., 2002. Tuberculosis—the global view. Journal of Medicine 346, T Rifampicin 0.2 0.0 0.0 (S) 1434–1435. T Isoniazid 2.0 4.0 0.0 (S) Chamundeeswari, D., Vasantha, J., Gopalakrishnan, S., 2004. Antibacterial and a Minimum inhibitory concentration. antifungal activities of Trewia polycarpa roots. Fitoterapia 75, 85–88. b DGI value (meanTSD) of the control vial was 20T1.4 for the sensitive Chandrasekaran, M., Venkatesalu, V., 2004. 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Journal of Ethnopharmacology 78, 213–216. tract infections may be due in part to a stimulation of the Lall, N., Das Sarma, M., Hazra, B., Meyer, J.J.M., 2003. Antimycobacterial immune system. activity of diospyrin derivatives and a structural analogue of diospyrin S.P.N. Mativandlela et al. / South African Journal of Botany 72 (2006) 232–237 237

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